Accuracy of serum luteinizing hormone and serum testosterone measurements to assess the efficacy of medical castration in prostate cancer patients
Morote et al. Journal of Biomedical Science
Accuracy of serum luteinizing hormone and serum testosterone measurements to assess the efficacy of medical castration in prostate cancer patients
Juan Morote 0 2
Imma Comas 1
Roser Ferrer 1
Jacques Planas 0 2
Anna Celma 0 2
Lucas Regis 0 2
0 Department of Urology, Vall d'Hebron Hospital, Universidad Autonoma de Barcelona , 14 Po Vall d'Hebron 119-129, 00173 Barcelona , Spain
1 Department of Biochemistry, Vall d'Hebron Hospital, Universitat Autònoma de Barcelona , Barcelona , Spain
2 Department of Urology, Vall d'Hebron Hospital, Universidad Autonoma de Barcelona , 14 Po Vall d'Hebron 119-129, 00173 Barcelona , Spain
Background: Luteinizing hormone-releasing hormone (LH-RH) agonists are the standard for androgen deprivation therapy (ADT) in prostate cancer (PCa) patients. Current guidelines recommend serum testosterone measurement to assess the efficacy of ADT and to define castration resistance. However, serum testosterone does not reflect the exclusive effect of castration due to its extratesticular production. The aim of this study is to analyze if serum LH reflects better than serum testosterone the activity of LH-RH agonists. Methods: Serum LH and serum testosterone were measured with chemiluminescent immunoassay (CLIA) in a cohort study of 1091 participants: 488 PCa patients “on LH-RH agonists”, 303 “off LH-RH agonist” in whom LH-RH agonists were withdrawn, and 350 men with PCa suspicion “no LH-RH agonist” who never received LH-RH agonists. In a validation cohort of 147 PCa patients, 124 on “LH-RH agonists” and 19 “off LH-RH agonists”, serum testosterone was also measured with liquid chromatography and tandem mass spectrometry (LC MSMS). Results: The area under the curve (AUC) to distinguish patients “on versus off LH-RH agonists” was 0.997 for serum LH and 0.740 for serum testosterone, P < 0.001. The 97.5 percentile of serum LH in patients “on LH-RH agonists” was 0.97 U/L, been the most efficient threshold 1.1 U/L. The AUCs for serum LH, testosterone measured with CLIA and with LC MSMS, in the validation cohort, were respectively 1.000, 0.646 and 0.814, P < 0.001. The efficacy to distinguish patients “on versus off LH-RH agonists” was 98.6%, 78.3%, and 89.5% respectively, using 1.1 U/L as threshold for serum LH and 50 ng/dL for serum testosterone regardless the method. Conclusions: Serum LH is more accurate than serum testosterone regardless the method, to distinguish patients “on versus off LH-RH agonists”. The castrate level of serum LH is 1.1 U/l. These findings suggest that assessment of LH-RH agonist efficacy and castration resistance definition should be reviewed.
Prostate cancer; Androgen deprivation therapy; LH-RH agonist; Testosterone; Luteinizing hormone
Medical castration with LH-RH agonists is currently the
standard of ADT in patients with PCa [
guidelines recommend measurements of testosterone in
serum or plasma to assess the efficacy of ADT and also
to define the castration resistance when biochemical or
clinical progression appears [
]. The current castration
level of serum testosterone is up to 50 ng/dl; however,
microelevations over this level are observed in up to
25% of PCa patients on LH-RH agonist [
]. The origin
of these microelevations of serum testosterone remains
unclear. They may be due to an inadequate castration or
a response to a new administration of LH-RH agonist.
An extratesticular over-production of testosterone in the
adrenal cortex or in the tumour cells may be also
]. These microelevations of serum
testosterone have been associated with poorer disease
The behaviour of the hypothalamic-pituitary-gonadal axis
varies according to the type of castration. After surgical
castration elevated levels of serum LH are present while
medical castration promptly induces a severe reduction in serum
LH due the complete blockade of pituitary gland receptors
]. After an acute LH-RH agonist administration an initial
over-production of serum LH precedes the maximal
blockade of pituitary LH receptors. This secondary flare of serum
testosterone may induce clinical consequences avoidable
with prophylactic administration of anti-androgens [
contrast, LH-RH antagonist administration rapidly reduces
serum LH and testosterone as surgical castration does [
The measurement of serum LH is sensible to assess the
effect of LH-RH agonists or antagonists [
The method to measure serum testosterone is crucial [
Nowadays chemiluminescent immunoassays (CLIAs) are
worldwide used because they are sensitive, automatable, fast
and cheap. However, due to their lack of accuracy and
reproducibility, they are not recommended especially to measure
low levels of testosterone [
]. The only recommended
methods to measure serum testosterone are those based on
previous chromatography and mass spectrometry [
Our hypothesis is that serum LH measurements are more
accurate than serum testosterone to assess the activity of
LH-RH agonists. The main objective of this study was to
demonstrate that serum LH correlates better than serum
testosterone with LH-RH agonist’s activity. Secondary
objectives were to define the castrate level of serum LH and
to validate previous results when serum testosterone was
measured with liquid chromatography and tandem mass
spectrometry (LC MSMS).
Design and participants
We prospectively measured serum LH and serum
testosterone in 1238 men divided into a study cohort of 1091
participants and a validation cohort of 147. In the study
cohort, 791 participants had histologically confirmed PCa
and received LH-RH agonist. A subset of 488 patients “on
LH-RH agonist” were on continuous treatment for longer
than 3 month, and 303 patients “off LH-RH agonists”
received LH-RH agonists for 24 to 36 months as a
neoadjuvant treatment to radiotherapy. In all of these
participants the activity of LH-RH agonist finished at least 1
month before the biochemical measurements. A control
group of 350 men “no LH-RH agonist” with similar age
was randomly selected from 864 men scheduled to
prostatic biopsy due to PCa suspicion. Men of this control
group never received LH-RH agonists or 5-alpha
reductase inhibitors. The validation cohort, where serum
testosterone was measured with CLIA and LC MSMS,
included 124 PCa patients “on LH-RH agonists” and 19
“off LH-RH agonists”.
Measurements of serum LH and testosterone
Blood collection took place between 8:00 and 10:00 am,
and serum was extracted from the samples. LH was
measured with CLIA using the automated platform
Advia-Centaur XPi® (Siemens Inc., NY). The lowest
level of quantification (LLOQ) was 0.12 U/L, and
according to the manufacturer the intra-assay
coefficient of variation ranged between 2,3% and 3.0%, and
the inter-assay coefficient of variation between 1,5%
and 2.9%. Testosterone was also measured with CLIA,
using the automated platform Advia-Centaur XPi®
(Siemens Inc., NY). The LLOQ was 10 ng/dL, and
according to the manufacturer, the intra-assay
coefficient of variation ranged between 2.3% and 6.2%, and
the inter-assay coefficient of variation between 2.7%
and 6.9% (Manufacturer information: 10629910_ES
Rev. U, 2014–08, 1–18). Testosterone was also measured
with LC MSMS in the validation cohort. An ultra
highpressure liquid chromatography with the 1290 Infinity
Binary LC System (Agilent Technologies, Santa, CA) was
performed. The system is connected in parallel to a
tandem mass spectrometry using the 6430 Series Tripe
Quadruple LC/MS System (Agilent Technologies, Santa
Clara, CA). The LLOQ was 2 ng/dL and thee intra-assay
coefficient of variation ranged from 4% to 5% and the
inter-assay coefficient of variation between 7% and 8%, at
levels of ST below 50 ng/dL [
The behaviours of serum LH and serum testosterone were
analysed in every group using the Shapiro test. The 2.5, 25,
50, 75, and 97.5 percentiles of serum LH and testosterone
were estimated and also after their logarithmic
transformation if no kurtotic distribution was observed [
Comparisons between two or more groups were carried out with the
Mann Witney U test and the Kruscal-Wallis test. ROC
curves were generated and areas under the curve (AUC)
were calculated and compared. The castration level of
serum LH was estimated from the study cohort, and
sensitivity, specificity and positive and negative predictive values
(PPV and NPV) were calculated. Up to 50 ng/dl was
considered the castration level of serum testosterone regardless of
the method used [
]. This analysis was carried out using the
Statistical Package for the Social Sciences (SPSS), version 20.
Serum LH and serum testosterone did not follow a
kurtotic distribution in any group, P < 0.001. The median
age was 70 years in patients “on LH-RH agonists”,
71 years in patients “off LH-RH agonists” and 70 years
in men “no LH-RH agonists”, P = 0.367. Statistics
analysing serum LH and serum testosterone levels in the
study cohort are summarized in Table 1. The median
serum LH was below the LLOQ in patients “on LH-RH
agonist”. In fact, the 75th percentile was below 0.12 U/L. In
contrast, the median of serum LH in the group of patients
“off LH-RH agonists” was higher than the observed in men
who never received an LH-RH agonist, P < 0.001. The
trend for serum testosterone was somewhat different. The
lowest level of testosterone was observed in patients “on
LH-RH agonists”. Patients “off LH-RH agonists” had a
moderately high level of serum testosterone, P < 0.001,
while a much higher amount was recorded in men who
never received LH-RH agonist, P < 0.001.
The ROC curves of serum LH and serum testosterone to
distinguish patients “on LH-RH agonist” from those “off or
no LH-RH agonists” are presented in Fig. 1. The AUCs of
serum LH were 0.998 and 0.997 respectively, while those of
serum testosterone were 0.993 and 0.740. The efficacy of
serum LH to distinguish patients “on LH-RH agonists”
from those “off or no LH-RH agonists” was significantly
higher than the observed for serum testosterone, P < 0.001.
The 97.5th percentile of serum LH in the subset of
patients “on an LH-RH agonist” was 0.85 U/L and
increased to 0.97 U/L after its logarithmic transformation.
However, the most efficient threshold of serum LH to
distinguish patients “on LH-RH agonists” from those “off or
no LH-RH agonists” was 1.1 U/L. The efficacy parameters
of 1.1 U/L of serum LH and 50 ng/dL of serum
testosterone are presented in Table 2. The castrate level of serum
LH distinguished 99.1% of patients while serum
testosterone 75.7%. The probability to be “on LH-RH agonist”
when serum LH was lower than 1.1 U/L was 99.8%, while
the likelihood of being “off or no LH-RH agonists” when
serum LH was higher than 1.1 U/L was 98.1%. The PPV
and NPV of serum testosterone were 78.5% and 75.1%,
The ROC curves of serum LH and serum testosterone
in the validation cohort are presented in Fig. 2. The
AUC of serum LH was 1.000, been 0.647 when serum
testosterone was measured with CLIA and 0.814 when it
was measured with LC MSMS, P < 0.001.
The parameters of efficacy for 1.1 U/L of serum LH
and 50 ng/dL for serum testosterone measured with
CLIA and LC MSMS are presented in Table 3. The
efficacy rates for distinguishing patients “on versus off
LHRH agonist” were 98.6%, 78.3% and 89.5%, respectively.
Serum LH PPV and NPV were 100% and 89.5%
respectively. Serum testosterone PPV and NPV were 90.6% and
23.1% when it was measured with CLIA and 91.1% and
62.5% respectively when it was measured with LC
Serum testosterone 408/488 (83.6) 191/303 (63.0)
aPPV Positive predictive value, bNPV Negative predictive value, cLH Luteinizing hormone
This is the first study to analyse the accuracy of serum
LH and serum testosterone as markers of medical
castration in patients undergoing LH-RH agonists. Our
results suggest that serum LH can better distinguish
patients on LH-RH agonist from those off LH-RH
agonist than serum testosterone does distinguish.
Under normal conditions, between one and 2% of
serum testosterone amount is synthesised in the adrenal
]. Evidence exists that testosterone may also
be produced by prostate cancer cells as well [
Microelevations of serum testosterone in patients
undergoing LH-RH agonists are therefore not always
secondary to an inadequate castration .
The acute injection of an LH-RH agonist prompts the
marked release of LH, whereas prolonged administration
produces inhibitory effects. Repeated injections of LH-RH
agonists and the use of depot preparations suppress the
function of pituitary gonadotrophs and the secretion of
]. As a consequence of this serum LH
behaviour following LH-RH agonist administration, serum
testosterone level increases due to a previous increase of
serum LH; decreasing to castration levels usually occurs
within 4 weeks after treatment [
]. The initial rise in
serum testosterone does not take place if LH-RH
antagonists are administered and castration levels of serum
testosterone are reached within a few hours, which is
similar to what happens after surgical castration [
The behaviour of serum testosterone and serum LH after
LH-RH agonist withdrawal has also been studied. The
overall impression is that serum testosterone recovery may be
slow, especially following a long administration of LH-RH
agonists, while serum LH quickly increases after LH-RH
withdrawal, reaching levels greater than those observed
before LH-RH agonist administration [
18, 20, 21
LH measurement has been used to analyse the switch from
LHRH antagonists to LH-RH agonists; it has been noted that
it remains low and stable during this manoeuvre [
The first objective of our study was to demonstrate that
serum LH correlates better than serum testosterone with
Serum testosterone (CLIA)d
Serum testosterone (LC MSMS)e 123/126 (97.6) 5/17 (29.4) 128/143 (89.5) 123/135 (91.1) 5/8 (62.5)
aPPV positive predictive value, bNPV negative predictive value, cLH Luteinizing hormone, dCLIA Chemiluminiscent immunoassay, eLC MSMS Liquid chromatography
tandem mass espectrometry
the active treatment of LH-RH agonists. The ROC curves
clearly indicate that the ability of serum LH to distinguish
between patients currently on LH-RH agonists and those
who are off LH-RH agonists is superior to that of serum
testosterone, even when it is measured using LC MSMS.
There is no information in the literature to compare our
results with. In our study we confirm, though, a similar
behaviour of serum testosterone when measured using
CLIA as we had observed in our previous studies [
Our second objective was to define the castration level
of serum LH in patients undergoing LH-RH agonist,
which had never been described before. We estimated
this level from the 97.5 percentile of serum LH
distribution in patients on LH-RH agonist, after its logarithmic
transformation. The overlap of serum LH values in
patients “on LH-RH” agonists and “off LH-RH” agonist
was also considered. We finally established in 1.1 U/l the
most efficient threshold, which was able to classify
adequately the 99.1% of patients in the study cohort and
the 98.6% in the validation cohort.
Finally, our tertiary objective was to validate the previous
results in a validation cohort of patients in whom serum
testosterone was measured simultaneously with CLIA and
LC MSMS as reference method [
]. Here, the ROC curves
reproduced similar results than those observed in the study
cohort. The effectiveness of serum LH to distinguish
patients “on versus off” LH-RH agonists was 98.6%, which
was significantly higher than the 78.3% and 89.5% observed
when testosterone was measured respectively with CLIA
and LC MSMS.
Our data indicated that serum LH is better marker of
medical castration than serum testosterone regardless
the method. A limitation of our study was the difficulty
to know the exact moment when a patient loss the effect
of LH-RH agonist, especially in those patients using
long-depot formulations. The behaviour of serum
testosterone after LH-RH agonist withdrawal is well
known trough the intermittent ADT studies [
however, less is known after long periods of continuous
LHRH agonist treatment [
]. Moreover, there is a lack of
knowledge about the exact behaviour of serum LH after
LH-RH agonist withdrawal because the actual CLIAs
were not always used in the past [
]. The dynamics of
serum LH after LH-RH agonist withdrawal remains
unclear and should be analysed in well-designed
prospective longitudinal studies.
Serum LH is better marker of medical castration than
serum testosterone is, regardless the method of
measurement. Serum LH is able to distinguish patients “on versus
off” LH-RH agonists with higher accuracy than serum
testosterone does. We propose 1.1 U/l as the castrate level of
serum LH, which is able to distinguish patients “on versus
off” LH-RH agonist activity with an efficacy similar to 99%.
Based on these findings, the assessment of LH-RH agonists
efficacy and especially the definition of castration resistance
should be redefined.
ADT: Androgen deprivation therapy; AUC: Area under the curve;
CLIA: Chemiluminiscent immunoassay; LC MSMS: Liquid chromatography
tandem mass spectrometry; LH: Luteinizing hormone; LH-RH: Luteinizing
hormone releasing hormone; LLOQ: Lowest level of quantification;
NPV: Negative predictive value; PCa: Prostate cancer; PPV: Positive predictive
value; ROC: Receiver operating characteristic
Availability of data and materials
JM and JP conceived design and basic concepts. IC and RF performed
laboratory determinations. AC, JP and LR provided patients data. JM
performed statistical analysis and wrote the article. All authors read and
approved the final version of the final manuscript.
Ethics approval and consent to participate
This study was approved by the ethical committee of Vall d’Hebron research
institute, (PR/AG 048–2016). Samples were collected after obtaining informed
consent from subjects and the methods were carried out in accordance with
relevant guidelines and regulations.
Consent for publication
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